CN115960913A - Application of tumorous stem mustard BjuA017664 gene in improving drought stress resistance of plants - Google Patents

Abstract

The invention discloses an application of a tumorous stem mustard BjuA017664 gene in improving drought stress resistance of plants, wherein the nucleotide sequence of the BjuA017664 gene is shown as SEQ ID NO.1, and the coded amino acid sequence is shown as SEQ ID NO. 2. The invention reports the application of the tumorous stem mustard BjuA017664 gene in enhancing the drought resistance of plants for the first time. Through constructing BjuA017664 gene overexpression vector, enabling the overexpression vector to be overexpressed in stem mustard and carrying out drought stress treatment, researches show that the normal growth condition is recovered after the drought stress treatment, the normal growth of transgenic arabidopsis can be greatly recovered, and the survival rate of transgenic lines reaches 75% or more and is obviously higher than that of wild plants. The over-expression of the tumorous stem mustard BjuA017664 gene is shown to be capable of obviously improving the function of drought stress resistance of plants. The gene provides a new gene target and resource for drought-resistant genetic improvement of crops, can be widely applied to genetic breeding, germplasm resource improvement and cultivation of plants, and has important significance for promoting stable yield of crops.

Description

Application of tumorous stem mustard BjuA017664 gene in improving drought stress resistance of plants

Technical Field

The invention belongs to the technical field of plant genetic engineering, and particularly relates to an application of a tumorous stem mustard BjuA017664 gene in improving drought stress resistance of plants.

Background

In recent years, with global warming and increased human activities, the frequency and number of extreme weather occurrences such as drought, flood, high temperature and frost have increased gradually, and the threat to agricultural production has become severe. Drought, high temperature, is currently considered to be two major abiotic stress factors that cause crop losses and outages. The drought stress of the plants due to water shortage can be caused by over-dry outside air or low soil water content, and the drought stress has a serious inhibiting effect on the growth and development of the plants.

Drought, one of the main limiting factors in the plant growth process, can block the respiration, photosynthesis and pore movement of plants, thereby affecting the growth and development and physiological metabolism of plants. Therefore, the method has important significance for mining drought-resistant related genes, exploring the drought-resistant mechanism of the genes and cultivating new drought-resistant varieties and ensuring the stability and safety of crops in China. In recent years, a great deal of research is carried out on the mechanism of plants responding to adversity stress such as drought and the like from the aspects of physiology, biochemistry, metabolism, ecology, heredity, evolution and the like, and due to the complexity of drought resistance characters of the plants, the drought resistance of the plants is difficult to be improved by adopting a traditional breeding method. With the development of molecular biology, people can know the stress resistance mechanism of plants to drought stress at molecular levels of gene composition, expression regulation, signal conduction and the like, and a new way is developed for improving the stress resistance of plants by using genetic engineering means. Some progress has been made in the study of genetic transformation of plants, so that the drought resistance of plants can be improved by a transgenic method. Transgenosis is an important means for plants to control their protein content by molecular means under stress conditions. The method has the advantages of high breeding speed, easy acquisition of resistant plants, better genetic stability and the like, and becomes a main means for acquiring the resistant plants. Therefore, the improvement of the drought resistance of crops by using a genetic engineering means and the improvement of the adaptability of crops and economic crops to stress are key problems and major problems which need to be solved urgently in new variety cultivation, but the separation of efficient drought resistance genes becomes a main factor for limiting the stress resistance genetic engineering of plants.

Stem tumor mustard (Brassica juncea var. Tubida Tsen et Lee), also known as cabbage head, is a Brassica plant of cruciferae, is a main raw material of tuber mustard, and is one of main economic crops in Chongqing, sichuan, zhejiang and other areas of China. For many years, the research on tumorous stem mustard mainly focuses on the fields of genetic breeding, improved variety breeding, cultivation technology, quality safety and the like, and the research on biology is continuously reported only in recent years. Previous studies have shown that tumorous stem mustard has a strong resistance to abiotic stress, and a plurality of adversity stress-related genes have been obtained from tumorous stem mustard at present. However, the drought-resistant genes obtained so far are too few to meet the needs of molecular design for improving plant yield and quality, so that the application of the tumorous stem mustard drought-resistant genetic engineering in agricultural production and commerce is greatly limited. Therefore, there is still a need to obtain a highly efficient, broad-spectrum drought-resistant gene in tumorous stem mustard.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide the application of the tumorous stem mustard BjuA017664 gene in improving the drought stress resistance of plants, provide a new gene target for improving the drought-resistant varieties of crops and enrich a gene bank.

In order to achieve the purpose, the invention adopts the following technical scheme: an application of a tumorous stem mustard BjuA017664 gene in improving drought stress resistance of plants, wherein the nucleotide sequence of the BjuA017664 gene is shown as SEQ ID NO.1 or has a nucleotide sequence with the same function obtained by replacing, deleting or inserting one or more nucleotides into the nucleotide sequence shown as SEQ ID NO. 1.

Furthermore, the amino acid sequence coded by the BjuA017664 gene is shown in SEQ ID NO.2 or has the amino acid sequence with the same function obtained by replacing, deleting or inserting one or more amino acids in the amino acid sequence shown in SEQ ID NO. 2.

The invention also provides a biological material containing the tumorous stem mustard BjuA017664 gene, wherein the biological material is recombinant DNA, an expression cassette, a transposon, a plasmid vector, a viral vector or an engineering bacterium.

The invention also provides application of the biological material in improving drought stress resistance of plants.

Further, the plant is a dicotyledonous plant or a monocotyledonous plant, preferably stem tumor mustard, arabidopsis thaliana, chinese cabbage, tobacco or rape.

Another object of the present invention is to provide a method for improving drought stress resistance of a plant, comprising: improving the expression quantity and/or activity of BjuA017664 protein in plants; the amino acid sequence of the BjuA017664 protein is shown as SEQ ID NO.2 or has the amino acid sequence which is obtained by replacing, deleting or inserting one or more amino acids of the amino acid sequence shown as SEQ ID NO.2 and has the same function.

Further, the plant is a dicotyledonous plant or a monocotyledonous plant, preferably stem tumor mustard, arabidopsis thaliana, chinese cabbage, tobacco or rape.

Further, the method specifically comprises the following steps: transforming the prepared or provided expression vector containing the BjuA017664 gene into agrobacterium to obtain agrobacterium engineering bacteria, then impregnating the agrobacterium engineering bacteria with a stem tumor mustard explant, and performing overexpression on the BjuA017664 gene by adopting a hypocotyl genetic transformation technology to obtain the drought stress resistant transgenic plant.

Compared with the prior art, the invention has the following beneficial effects:

1. the invention reports the application of the tumorous stem mustard BjuA017664 gene in enhancing the drought resistance of plants for the first time. Through constructing BjuA017664 gene overexpression vector, enabling the overexpression vector to be overexpressed in stem mustard and carrying out drought stress treatment, researches show that the normal growth condition is recovered after the drought stress treatment, the normal growth of transgenic arabidopsis can be greatly recovered, and the survival rate of transgenic lines reaches 75% or more and is obviously higher than that of wild plants. The result shows that the tumorous stem mustard BjuA017664 gene can be introduced into crops by biological means such as transgenosis and the like and is over-expressed, so that the drought stress resistance of the plants can be obviously improved, and the tumorous stem mustard BjuA017664 gene is a high-quality candidate gene for cultivating drought-resistant crop varieties.

2. The tumorous stem mustard BjuA017664 gene can obviously enhance the drought resistance of plants, is beneficial to maintaining the yield of crops under the drought stress condition, can reduce the loss of the drought stress on economic crops, and has obvious economic value. In addition, the invention also provides new gene targets and resources for the drought resistance genetic improvement of crops, can be widely applied to genetic breeding of plants, germplasm resource improvement and cultivation, and has important significance for promoting the stable yield of crops.

Drawings

FIG. 1 is a graph of the semi-quantitative expression analysis of the tumorous stem mustard BjuA017664 gene in over-expressed transgenic lines; WT was a wild-type tumorous stem mustard plant, and #2, #3, #4 and #5 were transgenic tumorous stem mustard plants, respectively.

FIG. 2 is a phenotype graph of a transgenic line overexpressing BjuA017664 during drought stress; WT was a wild-type tumorous stem mustard strain and #2, #3, #4 and #5 were transgenic tumorous stem mustard strains, respectively.

FIG. 3 is the survival rate after drought stress of transgenic lines overexpressing BjuA 017664; WT was a wild-type tumorous stem mustard strain, and #2, #3, #4 and #5 were transgenic tumorous stem mustard strains, respectively.

Detailed Description

The present invention will be described in further detail with reference to the following specific embodiments and the accompanying drawings. In the examples, the raw materials are ordinary commercial products unless otherwise specified. The experimental procedures described in the examples are not specifically described, i.e., they are carried out according to conventional molecular biological experimental procedures.

EXAMPLE 1 cloning of the tumorous stem mustard BjuA017664 Gene sequence

To validate the full-length gene of the stem tumor mustard BjuA017664, specific primers SEQ ID NO.3 (BjuA 017664-F) and SEQ ID NO.4 (BjuA 017664-R) were designed for PCR amplification analysis.

Taking leaf tissue of tumorous stem mustard in bud stage as material, and adopting TRIzol ^TM Plus RNA Purification Kit(12183555，Invitrogen ^TM ) Total RNA was extracted according to the protocol using DNase I (18047019, invitrogen ^TM ) Residual traces of DNA were removed and the concentration of RNA was determined spectrophotometrically and stored.

Approximately 2.0. Mu.g of total RNA from leaf stem tumor Arabidopsis thaliana was used to synthesize first strand cDNA according to the PrimeScript II first-strand cDNA synthesis kit (6210A, takara) protocol.

The PCR amplification system is high fidelity amplification enzyme Prime STAR HS (R010A, taKaRa) 0.25 mu L,5XPrimeSTAR Buffer (Mg) ²⁺ Plus) 5. Mu.L, forward primer (Bju-F, 10. Mu.M) 0.5. Mu.L, reverse primer (Bju-R, 10. Mu.M) 0.5. Mu.L, template (DNA) 1. Mu.L, dNTP (2.5 mM) 2. Mu.L, sterile ddH ₂ O make up to 25. Mu.L.

The sequences of the forward and reverse primers are shown below:

BjuA017664-F：ATGGCTACCGGAGAGGAGAAACCTG

BjuA017664-R：TCAGTGCTTGATCTTAGGCTTCTTC

the PCR reaction program is: pre-denaturation at 95 deg.C for 5min; at 95 ℃ for 30s; at 58 ℃ for 30s; 30s at 72 deg.C for 35 cycles; 72 ℃ for 10min.

The obtained PCR product is analyzed by agarose gel electrophoresis, and a specific amplification band can be observed at about 500bp under the irradiation of ultraviolet light. Purified according to the gel recovery kit (9672, takara) for use.

The purified DNA fragment was added with A using blunt-ended reagent and ligated with pMD20-T vector (6019, takara) by TA cloning, the ligation product was transformed into E.coli DH5a, and positive clones were picked from LB plates containing ampicillin (100 mg/L) and sequenced, which revealed that the CDS sequence of the Arabidopsis thaliana BjuA017664 gene was shown in SEQ ID No.1 and that it contained 489bp open reading frame, and the encoded protein contained 162 amino acids (shown in SEQ ID No. 2).

Example 2 construction and transformation of recombinant expression vector pTF101-BjuA017664-GFP

(1) Construction of pTF101-BjuA017664-GFP expression vector

According to the full-length gene sequence SEQ ID NO.1 of the tumorous stem mustard BjuA017664, primers Bju-gfp-F and Bju-gfp-R are designed, and enzyme cutting site sequences are introduced into the primers. Specific amplification of gene BjuA017664 was performed using the TA-ligated positive clone plasmid of example 1 as a template and Bju-gfp-F (forward primer) and Bju-gfp-R (reverse primer) as primers.

The primer sequences are as follows:

Bju-gfp-F：CCCGGGATGGCTACCGGAGAGGAGAAACCTG

Bju-gfp-R：GGATCCTCAGTGCTTGATCTTAGGCTTCTTC

wherein, the underlined sequence of Bju-gfp-F is SmaI restriction enzyme cutting site, and the underlined sequence of Bju-gfp-R is BamHI restriction enzyme cutting site.

And (3) PCR reaction system: hi-Fi Amplifier PrimeSTAR HS (R010A, taKaRa) 0.5. Mu.L, 5xPrimeSTAR Buffer (Mg) ²⁺ Plus) 10. Mu.L, forward primer (10. Mu.M) 1. Mu.L, reverse primer (10. Mu.M) 1. Mu.L, template (50-fold diluted plasmid) 1. Mu.L, dNTP (2.5 mM) 4. Mu.L, sterile ddH ₂ O make up to 50. Mu.L.

And (3) PCR reaction conditions: pre-denaturation at 95 deg.C for 5min; at 95 ℃ for 30s; at 58 ℃ for 30s; 30s at 72 deg.C for 35 cycles; 72 ℃ for 10min.

And (3) carrying out agarose gel electrophoresis detection on the PCR amplification product. The amplified target fragment has the same size as the expected fragment, and is recovered and purified according to the instructions of gel recovery kit (9672, takara), thus obtaining the target gene fragment.

The pTF101-GFP expression vector was digested with SmaI and BamHI. The enzyme cutting system is as follows: 5 mu L of pTF101-GFP vector; smaI 0.5 μ L; bamHI 0.5. Mu.L; buffer 10XK 2. Mu.L; sterile ddH ₂ O is complemented to 20 mu L; react at 37 ℃ for 3h. After completion of the digestion, the pTF101-GFP vector fragment was recovered according to the Takara agarose gel recovery kit.

T4 DNA Ligase (FL 101, trans) was used to construct pTF101-BjuA017664-GFP expression vector.

The connection reaction system is as follows:

50ng of purified PCR fragment (recovered BjuA017664 target fragment); 100ng of linear vector (pTF 101-GFP vector); 2 mu.L of 5xT4 DNA Ligase Buffer; 0.5 mu L of T4 DNA Ligase; sterile ddH ₂ O is complemented to 10 mu L; the reaction was carried out at 25 ℃ for 30min. The recombinant reaction system was transformed into E.coli DH5a according to the molecular cloning protocol and plated to a strain containing spectinomycin resistance(75 mg/L) screening culture plate, and positive clone sequencing to obtain the correct recombinant expression vector pTF101-BjuA017664-GFP containing BjuA017664 gene fragment. After the reporter gene GFP in the recombinant expression vector is fused with the 5' end of the target gene BjuA017664, the reporter gene GFP is positioned at the downstream of a constitutive promoter P35S to form fusion expression; the 3' end of BjuA017664 is assembled with NOS terminator, which can effectively terminate the transcription of fusion gene. The reporter gene GFP can emit green fluorescence without auxiliary factors and substrates after being excited by blue light, and can detect the expression condition of a target gene when being used as the reporter gene.

(2) Agrobacterium mediated genetic transformation of tumorous stem mustard

The constructed recombinant expression vector pTF101-BjuA017664-GFP was transferred into Agrobacterium strain GV3101 by a conventional freeze-thaw method, and positive clones were screened by PCR. Agrobacterium carrying the pTF101-BjuA017664-GFP vector was then introduced into tumorous mustard using a tumorous mustard hypocotyl stable genetic transformation technique. The expression levels of BjuA017664 genes in wild type and the transgenic strains (# 2, #3, #4 and # 5) which have good phenotype and overexpress BjuA017664 are identified by semi-quantitative RT-PCR. Trizol reagent (Invitrogen) was used ^TM ) Total RNA of leaf of Arabidopsis thaliana was extracted according to the procedures described and DNase I (Invitrogen) was used ^TM ) Residual DNA was removed and first strand cDNA was synthesized using cDNA reverse transcription reagent (Takara) and following the procedure described.

The detection target gene primer is as follows:

BjuA017664-F：ATGGCTACCGGAGAGGAGAAACCTG

BjuA017664-R：TCAGTGCTTGATCTTAGGCTTCTTC

the Arabidopsis thaliana internal reference primer is as follows:

RT-AtACTIN3-F：GGCTACTCTTTCACCACGAC

RT-AtACTIN3-R：GGATACCAGCATTCTCCATAC

as shown in FIG. 1, the expression of the target gene BjuA017664 was up-regulated in 4 transgenic lines (# 2, #3, #4 and # 5), while the expression of BjuA017664 was hardly detected (too low expression) in the wild-type plant (WT), indicating that BjuA017664 had been introduced into the genome of Arabidopsis and successfully transcribed.

Example 3 phenotypic Observation and analysis of transgenic tumorous Arabidopsis

Seeds of wild-type WT seeds and obtained transgenic lines #2, #3, #4 and #5 were respectively sterilized, sterilized and vernalized, germinated on water-soaked filter paper, seedlings 4 days after germination were transferred to a flowerpot (vermiculite: nutrient soil = 3:1) containing culture medium which was water-imbibed to saturation, placed in a plant culture room for culture, the culture room photoperiod being daytime: night = 1697 h, 8h, temperature 22 ℃. When the seedling plants all grow to 3 pairs of leaves, respectively carrying out drought stress (namely, not watering) treatment on the stem tumor mustard transgenic plant and the wild-type stem tumor mustard plant for 8 days, observing the growth state of the plants, analyzing the phenotype, then watering to carry out rehydration treatment on the plants, observing and counting the number of the plants recovering normal growth in each group after 2 days, and calculating the survival rate. The transgenic plants and wild-type plants were subjected to 3 times of repeated experiments, and 25 plants were subjected to stress treatment in each experimental group, and the results are shown in fig. 2 and 3.

The results show that before drought treatment, the transgenic lines overexpressing BjuA017664 and the wild type both grew normally with no significant difference (FIG. 2 a). After drought treatment, wild-type stems were completely unable to stand up, and the leaves of transgenic lines overexpressing BjuA017664 were wilted but still mostly able to stand up (FIG. 2 b). After rehydration, only a few wild plants are recovered to be normal, most of the transgenic material plants are recovered to be normal (figure 2 c), and through calculation, about 75 percent or more of BjuA017664 transgenic tumorous stem mustard strains recover to be normal in growth, wherein the transgenic #3 strains recover to be normal in growth, and the survival rate is about 100 percent; while only about 25% of the plants survived wild type tumorous stem mustard (fig. 3). The result shows that the over-expression of the tumorous stem mustard BjuA017664 gene can obviously improve the drought resistance of plants.

The sequences described in the above examples are specifically as follows:

sequence 1: SEQ ID NO.1BjuA017664 nucleotide sequence with length of 489bp

ATGGCTACCGGAGAGGAGAAACCTGTTATGGTCGTCGGTATGGACGAAAGCGAGCAGAGCACTTACGCCTTGGAGTGGACCCTCGATCGTTTCTTCGCTCCTTACGCTCCTAATTTTCCTTTCAAGCTCTTCATCGTCCACGCCAAACCTAACGCCGTCTCTGCCGTTGGTCTTGCTGGTCCCGGAGCTGCGGAGGTTCTGCCGTATGTTGATACCGATTTGAAGCATATTGCTGCCAGGGTTATCGAGATGGCTAAAGGTATCTGTCAGAGCAAATCGGTTGATGGCGCTATGTTCGAAGTTTTTGAAGGTGATGCAAGAAGTATACTGTGCGATGTTGTGGATAAACACCATGCTTCTCTTCTTGTCGTGGGAAGCCATGGTTATGGAGCTATCAAGAGGGCGGTTCTAGGGAGCGTGAGCGACTACTGTGCTCATCATGCTCATTGCTCGGTGATGATCGTGAAGAAGCCTAAGATCAAGCACTGA

Sequence 2: SEQ ID NO.2 protein BjuA017664 amino acid sequence, 162 amino acids long

MATGEEKPVMVVGMDESEQSTYALEWTLDRFFAPYAPNFPFKLFIVHAKPNAVSAVGLAGPGAAEVLPYVDTDLKHIAARVIEMAKGICQSKSVDGAMFEVFEGDARSILCDVVDKHHASLLVVGSHGYGAIKRAVLGSVSDYCAHHAHCSVMIVKKPKIKH*

And (3) sequence: SEQ ID NO.3BjuA017664-F nucleotide sequence with length of 25bp

ATGGCTACCGGAGAGGAGAAACCTG

And (3) sequence 4: SEQ ID NO.4BjuA017664-R nucleotide sequence with length of 25bp

TCAGTGCTTGATCTTAGGCTTCTTC

And (5) sequence: SEQ ID NO.5RT-AtACTIN3-F nucleotide sequence, length 20bp

GGCTACTCTTTCACCACGAC

And (3) sequence 6: SEQ ID NO.6RT-AtACTIN3-R nucleotide sequence, length 21bp

GGATACCAGCATTCTCCATAC

And (3) sequence 7: SEQ ID NO.7Bju-gfp-F nucleotide sequence, length 31bp

CCCGGGATGGCTACCGGAGAGGAGAAACCTG

And (2) sequence 8: SEQ ID NO.8Bju-gfp-R nucleotide sequence with length of 31bp

GGATCCTCAGTGCTTGATCTTAGGCTTCTTC

Finally, the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered in the claims of the present invention.

Claims (10)

1. An application of a tumorous stem mustard BjuA017664 gene in improving the drought stress resistance of plants, wherein the nucleotide sequence of the BjuA017664 gene is shown as SEQ ID No.1 or has a nucleotide sequence which is obtained by replacing, deleting or inserting one or more nucleotides in the nucleotide sequence shown as SEQ ID No.1 and has the same function.

2. The use of claim 1, wherein the BjuA017664 gene encodes an amino acid sequence as shown in SEQ ID No.2 or has an amino acid sequence with the same function obtained by substitution, deletion or insertion of one or more amino acids of the amino acid sequence as shown in SEQ ID No. 2.

3. A biomaterial containing the tumorous stem mustard BjuA017664 gene of claim 1, which is recombinant DNA, expression cassette, transposon, plasmid vector, viral vector or engineered bacterium.

4. Use of the biomaterial of claim 3 for improving drought stress resistance in plants.

5. The use of claim 1, 2 or 4, wherein the plant is a dicotyledonous plant or a monocotyledonous plant.

6. The use according to claim 5, wherein the plant is tumorous stem mustard, arabidopsis thaliana, chinese cabbage, tobacco or rape.

7. A method of increasing drought stress resistance in a plant comprising: improving the expression quantity and/or activity of BjuA017664 protein in plants; the amino acid sequence of the BjuA017664 protein is shown in SEQ ID NO.2 or has the amino acid sequence which is obtained by replacing, deleting or inserting one or more amino acids in the amino acid sequence shown in SEQ ID NO.2 and has the same function.

8. The method of claim 7, wherein the plant is a dicot or a monocot.

9. The method of claim 8, wherein the plant is tumorous stem mustard, arabidopsis thaliana, bok choy, tobacco or canola.

10. The method according to claim 7, characterized in that it comprises in particular the steps of: transforming agrobacterium with the prepared or provided expression vector containing the BjuA017664 gene as claimed in claim 1 to obtain agrobacterium engineering bacteria, and then infecting plants with the agrobacterium engineering bacteria to over-express the BjuA017664 gene to obtain drought stress resistant transgenic plants.

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